Herpes Simplex Viruses Type 1 and 2 (HSV-1 and HSV-2) are extremely prevalent and infect most people in the United States. These viruses cause lifelong, latent infections and can cause diseases from minor skin lesions to encephalitis. Unfortunately, preventative and therapeutic treatment options are limited. Networks of tegument proteins between the HSV-1 genomic capsid and envelope are likely to regulate many steps of the infection mechanism. A major gap in the progression of herpesvirology is the fact that the multiple specific functions of tegument proteins are largely unknown. Characterizing the many functions of these proteins is crucial to a complete understanding of the viral lifecycle. This proposal focuses on capsid-associated tegument protein UL21 that acts in the cytoplasm to regulate budding but is also found in the nucleus during infection. The objective of this proposal is to identify the multiple functions of HSV-1 UL21. The central hypothesis of this proposal is that UL21 has novel and unexplored nuclear functions in HSV-1 related to its ability to bind nucleic acids and localize to the nuclear rim. The hypothesis will be examined in two Specific Aims: Aim 1 will assess the binding of UL21 to nucleic acids and membranes to describe the mechanisms by which UL21 may regulate gene expression and nuclear egress, respectively. Furthermore, direct transcriptional effects of UL21 on UL16, another viral tegument protein, will be assayed in E coli. In Aim 2, the crystal structure of UL21 and its C- terminal domain will be solved and inspected for potential functional regions by characterizing the protein surface and by searching for structural similarity in proteins of known function. This proposal is significant because it will elucidate important functions previously hidden in the poorly understood tegument layer and aid researchers in assembling a more complete model of the infection mechanism-a necessary step to design new therapeutic strategies.